Terminal restriction fragment (TRF) analysis is the original technique that was developed for determining telomere length and, hence, is often described as the “gold standard” method. In this procedure, genomic DNA is exhaustively digested using a cocktail of frequent cutting restriction enzymes that lack recognition sites in the telomeric and subtelomeric regions (and hence do not “cut” telomeric DNA). The intact telomeres from all chromosomes are then resolved, based on size, using agarose gel electrophoresis, with the telomeric fragments being visualized by either southern blotting or in-gel hybridization using a probe specific for telomeric DNA. The varying lengths of telomeres will present as a smear, with the size and intensity of the smear being assessed by comparison to a DNA ladder comprising known fragment sizes.

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